The objectives of the proposed research are to establish an in vitro system where neurons of the norepinephrine (NE)- containing nucleus locus coeruleus (LC) or the serotonin (5-HT)- containing mesencephalic raphe (MR) nuclei will be grown together with neurons of one of their target organs, the hippocampus. These monoamine nuclei maintain an extensive, yet selective pattern of innervation of large parts of the brain and exert several different physiological actions. The rules governing these innervation patterns and functions are not known. The ionic basis of monoamine action is only beginning to be understood. The tissue culture methodology offers an advantage not shared by other test systems (e.g. the slice preparation); neurons can be grown under controlled conditions and the development of connections among different neurone types be monitored with both anatomical and physiological methods. It is proposed to grow embryonic rat hippocampal neurons in dissociated monolayer form or in thin organotypical slices. Embryonic LC or MR will be grown in dissociated form or in small explant near the hippocampal neurons. The conditions for formation of connections between the two neuron groups will be optimized and the effects of various hormones and peptides on these connections examined. LC or MR neurons will be recorded and activated by electrical or chemical stimuli. Postsynaptic hippocampal neurons will be recorded intracellularly with single or two electrode current/voltage clamp systems. The possible modulation of voltage sensitive conductances by the monoamines will be examined. The pharmacology of the types of receptors activated by these connections will be analyzed as well. These studies are expected to contribute to the understanding of the rules governing monoamine action in the brain and to provide a convenient test system for the analysis of drugs presumed to act on monoamine systems in the brain.